In vitro processing of the human alkyl-dihydroxyacetonephosphate synthase precursor

Alkyl-dihydroxyacetonephosphate synthase, a peroxisomal enzyme involved in the biosynthesis of ether phospholipids, is synthesized with a cleavable N-terminal presequence containing the peroxisomal targeting signal type 2. The human alkyl-dihydroxyacetonephosphate synthase precursor produced in vitro or expressed in Escherichia coli could be processed to a lower molecular weight protein by incubation at 37 degrees C with a guinea pig liver fraction, enriched in mitochondria, lysosomes, and peroxisomes. This lower molecular weight protein was identified as the mature human alkyl-dihydroxyacetonephosphate synthase by radiosequencing, indicating that the processing protease is present in this organellar fraction. Characterization of the processing protease indicated that it is a cysteine protease with a pH optimum of 6.5. Furthermore, it was demonstrated that exogenously added pre-alkyl-dihydroxyacetonephosphate synthase was imported and processed in purified peroxisomes in vitro. Processing of alkyl-dihydroxyacetonephosphate synthase did not increase the activity of the enzyme. This indicates that the presence of the presequence does not affect the activity of the enzyme.